Histocompatibility of Light-Curing Composites Used in Pediatric Dentistry in Human Oral Fibroblast Cells

Statement of the Problem: Tooth-colored composites are used to repair caries lesions and other dental defects, particularly in anterior regions in children. Although a wide range of composites is using, little attention has been paid to the important indicators such as biological profiles or products released from these materials. Purpose: The current study aimed to compare the histocompatibility and cytotoxicity of light-curing resin used to repair children's teeth with different brands (3M, DenFil, and Opallis) in curing times of 20 and 40 seconds in human oral fibroblast cells (HGF1). Materials and Method: In this in vitro study, Three types of flow composites (3M, Opallis, and DenFil), all at A2 shade, were used. The composites were at 4×2mm with separate exposure times of 20 and 40 seconds. MTT test was used to determine the cytotoxicity of composites on oral fibroblast cells. This test is based on the conversion of tetrazolium bromide to a purple compound known as formazan that its color intensity can be evaluated using the ELISA. The higher intensity of the color reveals the higher survival rate of the mitochondria, which indicates less toxicity. One-way variance analysis and unpaired t-test were used to compare the cytotoxicity of each brand in two conditions of 20 and 40 seconds of curing. Statistical significance was considered when p< 0.05. Results: 3M and Opallis composites were significantly reduced vitality of cells compared to control group in both 20s and 40s curing status. While DenFil brand did not show marked cytotoxicity. In each brand, there are no significant deference between 20s and 40s curing times. Conclusion: Histocompatibility depends on the type of composite resin. In the current study, DenFil brand showed the highest histocompatibility, followed by 3M and Opallis.


Introduction
Tooth-colored composites are used to restore tooth defects and other dental defects, particularly in the anterior regions [1]. These materials, like other dental materials, in addition to having good physical and chemical properties, should have appropriate tissue compatibility and do not cause damage or toxicity. Moreover, they should not induce inflammation or immune response [2]. This feature has been given a high weight in approving a dental substance, for instance by American Dental Association (ADA) and Food and Drug Administration (FDA). When using these materials to restore dental cavities, especially for those that are near the gums, their contact with gums is almost unavoidable [3].
On the other hand, due to their long contact with adjacent tissues like periodontium, the probability of cytotoxicity due to the presence of different chemical compounds is high. Therefore, it is necessary to investigate the toxic effects of monomers in these substances [3].
Some in vitro studies reported that polymerized resins have no harmful effects on humans. Unfortunately, the oral environment is not appropriate for polymathic reactions of these substances, and some studies showed that even cured compounds release chemical compounds [4][5]. Among different methods developed for cytotoxicity evaluation, the dimethylthiazol-2-yl diphenyltetrazolium bromide (MTT) test is more appropriate due to its high speed and low cost as well as higher sensitivity [6]. Developed by Mossman, this test is based on investigating the effect of a compound on the survival of mitochondria, which is based on the conversion of a salt called tetrazolium bromide to a purple compound known as formazan by an enzyme (succinate dehydrogenase) that its color intensity can be evaluated using the ELISA [6]. The higher intensity of the color reveals the higher survival rate of the mitochondria, which indicates less toxicity [7][8].
Histocompatibility indicates the function ability of a material in certain conditions in the presence of an appropriate host response [9]. The need to use materials with histocompatibility indicates the necessity of cytotoxicity studies. Basically, in vitro studies are intended to evaluate cytotoxicity or genetic toxicity of substances [4,[9][10]. Bationo et al. [9] assessed cytotoxicity of dental and orthodontic light-cured composite resins (Clearfil ES-2, Clearfil ES Flow, Filtek Supreme XTE, Grengloo, Blugloo, Transbond XT, and Transbond LR) and reported that the cell viabilities were between 85 and 90%. In another study, Franz et al. [10] compared the cytotoxicity of packable and non-packable composites in one-way and bilateral curing and found that twoway curing composites had less cytotoxicity. Although a wide range of composites is available, little attention has been paid to the important indicators such as biological profiles or products released from these materials.
Due to the high demand for operative dentistry, it is necessary to investigate the histocompatibility of currently available materials.
The current study aimed to compare the histocompatibility and cytotoxicity of three types of light-curing resin used to repair children's teeth with different brands (i.e., 3M, DenFil, and Opallis) in curing times of 20 and 40 seconds in human gingival fibroblast (HGF1).

Composite's preparation
In this in vitro study, three types of flowable composites (3M, Opallis and DenFil), all at A2 shade, were used (Table 1).
To facilitate the use of composites and access to a standard and desirable size in terms of the quantity and weight used for composite restorations, each composite was placed separately in circled forms made of Teflon in dimensions of 2mm thick and 4 mm in diameter [11].
The required amount of light cure composites were placed into the desired mold and cured by using LED light curing unit (850mW/cm2,woodpecker, Guang Dong, China) in two separate times of 20 and 40 seconds (to investigate the effect of radiation time) with a distance of 2mm. Then, we used a Mylar matrix strip on the surface to limit oxygen inhibition [12]. To maintain sterile conditions, all stages of composite preparation were carried out in a laminar hood equipped with a UV lamp. Three samples were taken from each composite group, and each of the prepared samples was placed in a separate 96-plate well. Subsequently, 200μl of Dulbecco's Modified Eagle's Medium (DMEM) solution was added to the medium, followed by incubation for 24h at 37 o C (CO2%5). We also considered a control group containing only the medium.

Cell Preparation
HGF-1 cell line was purchased from Pastor Institute of Iran (Tehran), as prepared vials. All cells were transferred to the fresh culture medium (containing 10% FBS and penicillin-ester), followed by incubation for 48h at 37 o C. Then, the cells were counted by trypan blue staining. This color cannot breach live cells, but it can enter the dead cells and turn them blue, which helps to count the cells.

MTT Test
Initially, 10000 live cells were poured into each of the  [13]. Eventually, the cell viability percentage of samples was calculated according to the following equation [13]: Cell viability percentage = (Absorbance of each sample/ Absorbance of control) × 100

Statistical Analysis
Data were analyzed using SPSS version 16 software.
One-way variance analysis was used to compare the cytotoxicity of different brands. In addition, the unpaired t-test was used to compare cell cytotoxicity at 20 and 40 seconds of curing. Statistical significance was considered when p< 0.05.

Results
In this study, cytotoxicity and survival percentage of human oral fibroblast cells after exposure with three different composites (DenFil, Opallis, and 3M) were investigated at times of 20 and 40 seconds ( Table 2)   reactions. In addition, unbound monomers can induce bacterial growth, particularly the microorganisms, which are involved in the formation of dental caries [16].
Even completely light-cured, HEMA is not fully linked; a part could be released and therefore, an allergic reaction can occur. HEMA can be able to pass through the dentin tubules and end up in pulp tissue. Furthermore, the potential toxic reactions of various associated monomers seem to be greater than toxicity of each monomer when studied individually [9]. Although these findings cannot be generalized to dental clinics, careful use of these materials is necessary, particularly for children who are more sensitive compared to adults [14].
In this experiment, the MTT test was used to investigate the viability and activity of cells, which is the most common test used for evaluating cytotoxicity. It is worth noting that the sensitivity of this test is higher than other currently available tests [17].
The present study aimed to investigate monomer release after 24 hours of exposure. In addition, previous studies [18][19] showed that the highest cytotoxicity effect of composites is in the first 24 hours. Triton et al. [18] and Tell et al. [19] investigated self-cure composites used in orthodontics and showed that immediately after mixing and final polymerization, these composites had high toxicity, which decreases over time but still there are levels of toxicity.
The current study investigated the effect of radiation duration (20 and 40 seconds), and a negative association was found, which was not statistically significant. In addition, according to the findings, increased volume of the filler and declined size of filler particles can improve biocompatibility.
D'Souza et al. [5], in a study on the cytotoxicity of light cure composites, concluded that the compounds cured for 40 seconds had lower cytotoxicity than those cured for 20 seconds. This issue refers to the importance of light cure duration [5], which can be attributed to the lower conversion of monomer-to-polymer and more release of toxic monomers when curing time is insufficient.
Franz et al. [10] investigated the cytotoxic effect of packable and non-packable composites in one-way and bilateral curing and showed that two-way curing composites had less toxicity. The level of monomer release affects the cytotoxicity of composites, which depends on several factors like light curing time, light penetration power, radiation intensity, and concentration of light initiators. These factors contribute to the full polymerization of these composites [20].
The sufficiency of conversion rate of monomers has a crucial role in their biocompatibility. Caughman et al. [20] reported a negative association between cytotoxicity and monomer release with light curing duration. In addition, it is proved that increased filler content and decreased filler particle size is associated with improved biocompatibility [5]. which is based on determining the quantity of biochemical activity of cells and the activity of some cell enzymes [23], but the results of this study did not indicate cell death. Hence, further studies are needed to achieve results that are more definitive.
According to the findings, it is suggested that during clinical application of composites, contact with gingival tissue should be prevented as much as possible. Protective equipment such as rubber dam is useful to achieve this purpose. In addition, over-contouring and invasion of restorative composites into the gingival space should be avoided as much as possible [24].

Conclusion
This study showed that composite resins used in pediatric dentistry have different biocompatibility standards, which depends on composition and percentage of unbound monomers. In the current study, DenFil (microhybrid) showed the highest histocompatibility, followed by 3M and Opallis (Microfill).

Acknowledgment
This study was extracted from dentistry student thesis of